Biology subjects

Broz, A. K.

Publications and source records attributed to Broz, A. K..

15 recordsLinked to original sources

bioRxiv2025

The effects of rapid mitochondrial gene loss on organellar proteomes

Mitochondrial genomes retain only a tiny number of genes from their bacterial progenitors, including key components of protein translation machinery. The set of mitochondrially encoded tRNAs and ribosomal subunits is highly variable across angiosperms, with many examples of mitochondrial gene loss, replacement, and/or transfer to the nucleus. This dynamic history suggests large-scale remodeling of mitochondrial translation machinery in some lineages, but such conclusions are largely inferred from genomic sequence and protein targeting predictions. Here, we use proteomic (LC-MS/MS) analysis of purified mitochondria and chloroplasts from angiosperm species with major differences in mitochondrial gene content (Arabidopsis thaliana and Silene conica). Our analysis largely confirms the current understanding of subcellular localization for nuclear-encoded proteins involved in tRNA metabolism and ribosome function in A. thaliana, although some aminoacyl-tRNA synthetases (aaRSs) may have more specialized subcellular roles than previously thought. In contrast, S. conica has undergone extensive mitochondrial gene loss and numerous associated changes in the composition of its mitochondrial proteome, including apparent retargeting of aaRSs, replacement of ribosomal subunits, and loss of the glutamine amidotransferase (GatCAB) complex. Overall, this analysis illustrates how the complex network of molecular interactions necessary for mitochondrial translation are perturbed by gene loss, transfer, and replacement. Significance StatementPlant mitochondrial genomes exhibit exceptional diversity in gene content across species due to ongoing processes of gene loss, transfer, and functional replacement. However, our understanding of the consequences of these genomic changes on the mitochondrial proteome remain limited. Here, we compare organellar (mitochondrial and chloroplast) proteomes across species of flowering plants, revealing a large-scale rewiring of mitochondrial protein synthesis machinery has coincided with loss of tRNA and ribosomal protein genes from the mitochondrial genome.

molecular biology↗

bioRxiv2025

Plant MutS2 proteins function in plastid ribosome quality control

Tight regulation of chloroplast translation is essential for plant growth, development and environmental response. Active translation can result in stalling and collision of ribosomes, which have negative fitness consequences. However, the ways in which chloroplasts respond to these types of translational stressors remain unknown. Here, we identify two MutS2 proteins that act as critical players in plastid ribosome-associated quality control (RQC) in Arabidopsis. We found that both MutS2A and MutS2B are required to overcome specific antibiotic-induced ribosome stalling and collisions. Further, these proteins appear to be essential for tissue greening during de-etiolation, potentially due to increased translational demand during the transition from etioplast to chloroplast. Although bacterial homologs of MutS2 have been widely recognized for their role in regulating homologous recombination, we found only weak support for this function in Arabidopsis plastids. Therefore, these proteins, which are widely conserved among photosynthetic eukaryotes, appear to be central in the resolution of ribosome collisions and may play a critical role during times of increased translational demand.

plant biology↗

bioRxiv2025

Flipping the switch on some of the slowest mutating genomes: Direct measurements of plant mitochondrial and plastid mutation rates in msh1 mutants

Plant mitochondrial and plastid genomes have exceptionally slow rates of sequence evolution, and recent work has identified an unusual member of the MutS gene family ("plant MSH1") as being instrumental in preventing point mutations in these genomes. However, the eXects of disrupting MSH1-mediated DNA repair on "germline" mutation rates have not been quantified. Here, we used Arabidopsis thaliana mutation accumulation (MA) lines to measure mutation rates in msh1 mutants and matched wild type (WT) controls. We detected 124 single nucleotide variants (SNVs: 49 mitochondrial and 75 plastid) and 668 small insertions and deletions (indels: 258 mitochondrial and 410 plastid) in msh1 MA lines. In striking contrast, we did not find any organelle mutations in the WT MA lines, and reanalysis of data from a much larger WT MA experiment also failed to detect any variants. The observed number of SNVs in the msh1 MA lines corresponds to estimated mutation rates of 6.1{xi}10-7 and 3.2 {xi}10-6 per bp per generation in mitochondrial and plastid genomes, respectively. These rates exceed those of species known to have very high mitochondrial mutation rates (e.g., nematodes and fruit flies) by an order of magnitude or more and are on par with estimated rates in humans despite the generation times of A. thaliana being nearly 100-fold shorter. Therefore, disruption of a single plant-specific genetic factor in A. thaliana is suXicient to erase or even reverse the enormous diXerence in organelle mutation rates between plants and animals.

genetics↗

bioRxiv2024

Expansion of the MutS gene family in plants

The MutS gene family is distributed across the tree of life and is involved in recombination, DNA repair, and protein translation. Multiple evolutionary processes have expanded the set of MutS genes in plants relative to other eukaryotes. Here, we investigate the origins and functions of these plant-specific genes. Land plants, green algae, red algae, and glaucophytes share cyanobacterial-like MutS1 and MutS2 genes that presumably were gained via plastid endosymbiotic gene transfer. MutS1 was subsequently lost in some taxa, including seed plants, whereas MutS2 was duplicated in Viridiplantae (i.e., land plants and green algae) with widespread retention of both resulting paralogs. Viridiplantae also have two anciently duplicated copies of the eukaryotic MSH6 gene (i.e., MSH6 and MSH7) and acquired MSH1 via horizontal gene transfer - potentially from a nucleocytovirus. Despite sharing the same name, "plant MSH1" is not directly related to the gene known as MSH1 in some fungi and animals, which may be an ancestral eukaryotic gene acquired via mitochondrial endosymbiosis and subsequently lost in most eukaryotic lineages. There has been substantial progress in understanding the functions of MSH1 and MSH6/MSH7 in plants, but the roles of the cyanobacterial-like MutS1 and MutS2 genes remain uncharacterized. Known functions of bacterial homologs and predicted protein structures, including fusions to diverse nuclease domains, provide hypotheses about potential molecular mechanisms. Because most plant-specific MutS proteins are targeted to the mitochondria and/or plastids, the expansion of this family appears to have played a large role in shaping plant organelle genetics. One-Sentence SummaryPlants are distinguished from other eukaryotes by a functionally diverse complement of MutS proteins gained via a combination of gene duplication, endosymbiotic gene transfer, and horizontal gene transfer.

evolutionary biology↗

bioRxiv2024

Disruption of recombination machinery alters the mutational landscape in plant organellar genomes

Land plant organellar genomes have extremely low rates of point mutation yet also experience high rates of recombination and genome instability. Characterizing the molecular machinery responsible for these patterns is critical for understanding the evolution of these genomes. While much progress has been made towards understanding recombination activity in land plant organellar genomes, the relationship between recombination pathways and point mutation rates remains uncertain. The organellar targeted mutS homolog MSH1 has previously been shown to suppress point mutations as well as non-allelic recombination between short repeats in Arabidopsis thaliana. We therefore implemented high-fidelity Duplex Sequencing to test if other genes that function in recombination and maintenance of genome stability also affect point mutation rates. We found small to moderate increases in the frequency of single nucleotide variants (SNVs) and indels in mitochondrial and/or plastid genomes of A. thaliana mutant lines lacking radA, recA1, or recA3. In contrast, osb2 and why2 mutants did not exhibit an increase in point mutations compared to wild type (WT) controls. In addition, we analyzed the distribution of SNVs in previously generated Duplex Sequencing data from A. thaliana organellar genomes and found unexpected strand asymmetries and large effects of flanking nucleotides on mutation rates in WT plants and msh1 mutants. Finally, using long- read Oxford Nanopore sequencing, we characterized structural variants in organellar genomes of the mutant lines and show that different short repeat sequences become recombinationally active in different mutant backgrounds. Together, these complementary sequencing approaches shed light on how recombination may impact the extraordinarily low point mutation rates in plant organellar genomes.

genetics↗

bioRxiv2023

Chromosome-level genome assembly for the angiosperm Silene conica

The angiosperm genus Silene has been the subject of extensive study in the field of ecology and evolution, but the availability of high-quality reference genome sequences has been limited for this group. Here, we report a chromosome-level assembly for the genome of Silene conica based on PacBio HiFi, Hi-C and Bionano technologies. The assembly produced 10 scaffolds (one per chromosome) with a total length of 862 Mb and only [~]1% gap content. These results confirm previous observations that S. conica and its relatives have a reduced base chromosome number relative to the genuss ancestral state of 12. Silene conica has an exceptionally large mitochondrial genome (>11 Mb), predominantly consisting of sequence of unknown origins. Analysis of shared sequence content suggests that it is unlikely that transfer of nuclear DNA is the primary driver of this mitochondrial genome expansion. More generally, this assembly should provide a valuable resource for future genomic studies in Silene, including comparative analyses with related species that recently evolved sex chromosomes. SignificanceWhole-genome sequences have been largely lacking for species in the genus Silene even though these flowering plants have been used for studying ecology, evolution, and genetics for over a century. Here, we address this gap by providing a high-quality nuclear genome assembly for S. conica, a species known to have greatly accelerated rates of sequence and structural divergence in its mitochondrial and plastid genomes. This resource will be valuable in understanding the coevolutionary interactions between nuclear and cytoplasmic genomes and in comparative analyses across this highly diverse genus.

genomics↗

bioRxiv2023

PHOTOSYNTHESIS DRIVES RETENTION OF BACTERIAL-LIKE TRNA METABOLISM IN PLANT ORGANELLES

Eukaryotic nuclear genomes often encode distinct sets of translation machinery for function in the cytosol vs. organelles (mitochondria and plastids). This raises questions about why multiple translation systems are maintained even though they are capable of comparable functions and whether they evolve differently depending on the compartment where they operate. These questions are particularly interesting in plants because translation machinery, including aminoacyl-tRNA synthetases (aaRS), is often dual-targeted to the plastids and mitochondria. These organelles have different functions, with much higher rates of translation in plastids to supply the abundant, rapid-turnover proteins required for photosynthesis. Previous studies have indicated that plant organellar aaRS evolve more slowly compared to mitochondrial aaRS in eukaryotes that lack plastids. Thus, we investigated the evolution of nuclear-encoded organellar and cytosolic aaRS and tRNA maturation enzymes across a broad sampling of angiosperms, including non-photosynthetic (heterotrophic) plant species with reduced plastid gene expression, to test the hypothesis that translational demands associated with photosynthesis constrain the evolution of enzymes involved in organellar tRNA metabolism. Remarkably, heterotrophic plants exhibited wholesale loss of many organelle-targeted aaRS and other enzymes, even though translation still occurs in their mitochondria and plastids. These losses were often accompanied by apparent retargeting of cytosolic enzymes and tRNAs to the organelles, sometimes preserving aaRS-tRNA charging relationships but other times creating surprising mismatches between cytosolic aaRS and mitochondrial tRNA substrates. Our findings indicate that the presence of a photosynthetic plastid drives the retention of specialized systems for organellar tRNA metabolism. SignificanceThe process by which endosymbionts are integrated into a host and become organelles results in a combination of gene loss, transfer to the nucleus, and retention in the organellar genome. It is not clear why some endosymbiont-derived genes may be retained when a functional host counterpart exists whose gene product could simply be retargeted to the organelles. This study revealed that the photosynthetic activity in plant plastids may be responsible for retention of functionally redundant tRNA processing machinery, while mitochondria are more flexible regarding substitution with cytosolic-type enzymes. Therefore, functional constraint in the plastid is likely more important than in the mitochondria for shaping the evolution and retention of functionally redundant proteins that are dual targeted to both organelles.

plant biology↗

bioRxiv2022

Dynamics of organelle DNA segregation in Arabidopsis development and reproduction revealed with tissue-specific heteroplasmy profiling and stochastic modelling

Organelle DNA (oDNA) in mitochondria and plastids is vital for plant (and eukaryotic) life. Selection against damaged oDNA is mediated in part by segregation - the sorting of different oDNA types into different cells in the germline. Plants segregate oDNA very rapidly, with oDNA recombination protein MutS Homolog 1 (MSH1), a key driver of this segregation, but in contrast to mammals, we have very limited knowledge of the dynamics of this segregation within plants and between generations. Here, we combine stochastic modelling with tissue-specific heteroplasmy measurements to reveal the trajectories of oDNA segregation in Arabidopsis thaliana development and reproduction. We obtain and use new experimental observations of oDNA through development to confirm and refine the predictions of the theory inferred from existing measurements. Ongoing segregation proceeds gradually but continually during plant development, with a more rapid increase between inflorescence formation and the establishment of the next generation. When MSH1 is compromised, we show that the majority of observed segregation could be achieved through partitioning at cell divisions. When MSH1 is functional, mtDNA segregation is far more rapid than can be achieved through cell divisions; we show that increased oDNA gene conversion is a plausible mechanism quantitatively explaining this acceleration. We also discuss the support for different models of the plant germline provided by these observations.

plant biology↗

bioRxiv2022

Avoiding misleading estimates using mtDNA heteroplasmy statistics to study bottleneck size and selection

Mitochondrial DNA (mtDNA) heteroplasmy samples can shed light on vital developmental and genetic processes shaping mtDNA populations. The sample mean and sample variance of a set of heteroplasmy observations are typically used both to estimate bottleneck sizes and to perform fits to the theoretical "Kimura" distribution in seeking evidence for mtDNA selection. However, each of these applications raises problems. Sample statistics do not generally provide optimal fits to the Kimura distribution and so can give misleading results in hypothesis testing, including false positive signals of selection. Using sample variance can give misleading results for bottleneck size estimates, particularly for small samples. These issues can and do lead to false positive results for mtDNA mechanisms - all published experimental datasets we re-analysed, reported as displaying departures from the Kimura model, do not in fact give evidence for such departures. Here we outline a maximum likelihood approach that is simple to implement computationally and addresses all of these issues. We advocate the use of maximum likelihood fits and explicit hypothesis tests, not fits and Kolmogorov-Smirnov tests via summary statistics, for ongoing work with mtDNA heteroplasmy.

genetics↗

bioRxiv2022

Sorting of plant mitochondrial and plastid heteroplasmy is extremely rapid and depends on MSH1 activity

The fate of new mitochondrial and plastid mutations depends on their ability to persist and spread among the numerous organellar genome copies within a cell (heteroplasmy). The extent to which heteroplasmies are transmitted across generations or eliminated through genetic bottlenecks is not well understood in plants, in part because their low mutation rates make these variants so infrequent. Disruption of MutS Homolog 1 (MSH1), a gene involved in plant organellar DNA repair, results in numerous de novo point mutations, which we used to quantitatively track the inheritance of single nucleotide variants in mitochondrial and plastid genomes in Arabidopsis. We found that heteroplasmic sorting (the fixation or loss of a variant) was rapid for both organelles, greatly exceeding rates observed in animals. In msh1 mutants, plastid variants sorted faster than those in mitochondria and were typically fixed or lost within a single generation. Effective transmission bottleneck sizes for plastids and mitochondria were N {approx} 1 and 4, respectively. Restoring MSH1 function further increased the rate of heteroplasmic sorting in mitochondria (N {approx} 1.3), potentially due to its hypothesized role in promoting gene conversion as a mechanism of DNA repair, which is expected to homogenize genome copies within a cell. Heteroplasmic sorting also favored GC base pairs. Therefore, recombinational repair and gene conversion in plant organellar genomes can potentially accelerate the elimination of heteroplasmies and bias the outcome of this sorting process. Significance statementMitochondria and plastids play essential roles in eukaryotic life; thus, mutations in these organellar genomes can have severe consequences. In animals, early germline sequestration creates genetic "bottlenecks" providing cell-to-cell variance in mitochondrial mutations upon which selection can act. However, the dynamics of organellar mutations in plants and other organisms that lack early germline segregation remain unclear. Here, we show that sorting of mutations in plant organellar genomes proceeds very rapidly - much faster than in animals. In mitochondria, this process is accelerated by MSH1, a gene involved in recombination and repair of organellar genomes. This suggests that in plants, recombinational repair creates cell-to-cell variance in the frequency of organellar mutations, facilitating selection in the absence of a classical germline bottleneck.

plant biology↗

bioRxiv2020

Detecting de novo mitochondrial mutations in angiosperms with highly divergent evolutionary rates

Although plant mitochondrial genomes typically show low rates of sequence evolution, levels of divergence in certain angiosperm lineages suggest anomalously high mitochondrial mutation rates. However, de novo mutations have never been directly analyzed in such lineages. Recent advances in high-fidelity DNA sequencing technologies have enabled detection of mitochondrial mutations when still present at low heteroplasmic frequencies. To date, these approaches have only been performed on a single plant species (Arabidopsis thaliana). Here, we apply a high-fidelity technique (Duplex Sequencing) to multiple angiosperms from the genus Silene, which exhibits extreme heterogeneity in rates of mitochondrial sequence evolution among close relatives. Consistent with phylogenetic evidence, we found that S. latifolia maintains low mitochondrial variant frequencies that are comparable to previous measurements in Arabidopsis. Silene noctiflora also exhibited low variant frequencies despite high levels of historical sequence divergence, which supports other lines of evidence that this species has reverted to lower mitochondrial mutation rates after a past episode of acceleration. In contrast, S. conica showed much higher variant frequencies in mitochondrial (but not in plastid) DNA, consistent with an ongoing bout of elevated mitochondrial mutation rates. Moreover, we found an altered mutational spectrum in S. conica heavily biased towards ATGC transitions. We also observed an unusually low number of mitochondrial genome copies per cell in S. conica, potentially pointing to reduced opportunities for homologous recombination to accurately repair mismatches in this species. Overall, these results suggest that historical fluctuations in mutation rates are driving extreme variation in rates of plant mitochondrial sequence evolution.

genetics↗

bioRxiv2020

Migration through a major Andean ecogeographic disruption as a driver of genotypic and phenotypic diversity in a wild tomato species

The large number of species on our planet arises from the phenotypic variation and reproductive isolation occurring at the population level. In this study, we sought to understand the origins of such population-level variation in defensive acylsugar chemistry and mating systems in Solanum habrochaites - a wild tomato species found in diverse Andean habitats in Ecuador and Peru. Using Restriction-Associated-Digestion Sequencing (RAD-seq) of 50 S. habrochaites accessions, we identified eight population clusters generated via isolation and hybridization dynamics of 4-6 ancestral populations. Estimation of heterozygosity, fixation index, isolation by distance, and migration probabilities, allowed identification of multiple barriers to gene flow leading to the establishment of extant populations. One major barrier is the Amotape-Huancabamba Zone (AHZ) - a geographical feature in the Andes with high endemism, where the mountainous range breaks up into isolated microhabitats. The AHZ was associated with emergence of alleles for novel reproductive and acylsugar phenotypes. These alleles led to the evolution of self-compatibility in the northern populations, where alleles for novel defense-related enzyme variants were also found to be fixed. We identified geographical distance as a major force causing population differentiation in the central/southern part of the range, where S. habrochaites was also inferred to have originated. Findings presented here highlight the role of the diverse ecogeography of Peru and Ecuador in generating new, reproductively isolated populations, and enhance our understanding of the microevolutionary processes that lay a path to speciation.

plant biology↗

bioRxiv2020

Long-read transcriptome and other genomic resources for the angiosperm Silene noctiflora

The angiosperm genus Silene is a model system for several traits of ecological and evolutionary significance in plants, including breeding system and sex chromosome evolution, host-pathogen interactions, invasive species biology, heavy metal tolerance, and cytonuclear interactions. Despite its importance, genomic resources for this large genus of approximately 850 species are scarce, with only one published whole-genome sequence (from the dioecious species S. latifolia). Here, we provide genomic and transcriptomic resources for a hermaphroditic representative of this genus (S. noctiflora), including a PacBio Iso-Seq transcriptome, which uses long-read, single-molecule sequencing technology to analyze full-length mRNA transcripts and identify paralogous genes and alternatively spliced genes. Using these data, we have assembled and annotated high-quality full-length cDNA sequences for approximately 17,000 S. noctiflora genes and 27,000 isoforms. We demonstrated the utility of these data to distinguish between recent and highly similar gene duplicates by identifying novel paralogous genes in an essential protease complex. Further, we provide a draft assembly for the approximately 2.7-Gb genome of this species, which is near the upper range of genome-size values reported for diploids in this genus and three-fold larger than the 0.9-Gb genome of S. conica, another species in the same subgenus. Karyotyping confirmed that S. noctiflora is a diploid, indicating that its large genome size is not due to polyploidization. These resources should facilitate further study and development of this genus as a model in plant ecology and evolution.

genomics↗

bioRxiv2020

MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

Mitochondrial and plastid genomes in land plants exhibit some of the slowest rates of sequence evolution observed in any eukaryotic genome, suggesting an exceptional ability to prevent or correct mutations. However, the mechanisms responsible for this extreme fidelity remain unclear. We tested seven candidate genes involved in cytoplasmic DNA replication, recombination, and repair (POLIA, POLIB, MSH1, RECA3, UNG, FPG, and OGG1) for effects on mutation rates in the model angiosperm Arabidopsis thaliana by applying a highly accurate DNA sequencing technique (duplex sequencing) that can detect newly arisen mitochondrial and plastid mutations still at low heteroplasmic frequencies. We find that disrupting MSH1 (but not the other candidate genes) leads to massive increases in the frequency of point mutations and small indels and changes to the mutation spectrum in mitochondrial and plastid DNA. We also used droplet digital PCR to show transmission of de novo heteroplasmies across generations in msh1 mutants, confirming a contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within the mutS mismatch repair family that we find is also present outside of green plants in multiple eukaryotic groups (stramenopiles, alveolates, haptophytes, and cryptomonads), as well as certain bacteria and viruses. MSH1 has previously been shown to limit ectopic recombination in plant cytoplasmic genomes. Our results point to a broader role in recognition and correction of errors in plant mitochondrial and plastid DNA sequence, leading to greatly suppressed mutation rates perhaps via initiation of double-stranded breaks and repair pathways based on faithful homologous recombination.

genetics↗

bioRxiv2016

Linking gene expression to unilateral pollen-pistil reproductive barriers

Unilateral incompatibility (UI) is an asymmetric reproductive barrier that unidirectionally prevents gene flow between species and/or populations. UI is characterized by a compatible interaction between partners in one direction, but in the reciprocal cross fertilization fails, generally due to pollen tube rejection by the pistil. Although UI has long been observed in crosses between different species, the underlying molecular mechanisms are only beginning to be characterized. The wild tomato relative Solanum habrochaites provides a unique study system to investigate the molecular basis of this reproductive barrier, as populations within the species exhibit both interspecific and interpopulation UI. Here we used a transcriptomic approach to identify genes in both pollen and pistil tissues that may be probable key players in UI. We confirmed UI at the pollen-pistil level between a self-incompatible population and a self-compatible population of S. habrochaites. A comparison of gene expression between pollinated styles exhibiting the incompatibility response and unpollinated controls revealed only a small number of differentially expressed transcripts. Many more differences in transcript profiles were identified between UI-competent versus UI-compromised reproductive tissues. A number of intriguing candidate genes were highly differentially expressed, including a putative pollen arabinogalactan protein, a stylar Kunitz family protease inhibitor, and a stylar peptide hormone Rapid Alkalinization Factor. Our data also provide transcriptomic evidence that fundamental processes including reactive oxygen species signaling are likely key in UI pollen-pistil interactions between both populations and species. Our transcriptomic analysis highlighted specific genes, including those in ROS signaling pathways that warrant further study in investigations of UI. To our knowledge, this is the first report to identify candidate genes involved in unilateral barriers between populations of the same species.

plant biology↗